HPLC column technology these days is to a great extent inspired by monoliths [1-4]. Monoliths which are also called continuous polymer beds, continuous polymer rods or continuous column supports can be described as a single piece of porous polymer [5]. They have been shown to smooth out some significant limitations of microparticulate columns, mainly in terms of hydrodynamic properties [6] and separation efficiency towards biomolecules due to convective flow [7].
During the last 15 years, HPLC column fabrication based on monolithic materials has gained considerable attention. Inorganic (silica) monolithic polymer networks have been prepared by sol-gel process using silane-precursors starting in 1996 [8,9] and are nowadays commercially available in conventional HPLC as well as in capillary size format (Chromolith™, Merck, Darmstadt, Germany) [10]. Their silica skeleton is characterized by a bimodal pore-size distribution of gigapores or through pores (˜2 μm), enabling high flow at moderate back pressure, and mesopores (˜15 μm) providing high surface area [11]. This distribution of porosity offers improved resolution and speed of separation regarding small molecules. The analysis of biopolymers (especially biomolecules of high molecular size like proteins or dsDNA fragments), however, is limited due to insufficient presence of macropores [4].
The area of monoliths based upon polymerization of organic monomers can be divided into (meth)acrylate and styrene chemistry [12].
A great diversity of acrylates and methacrylates was employed for monolith fabrication. Next to thermally and chemically [13], also photochemically initiated free radical polymerization of UV-transparent monomers has proven to be suitable for HPLC column design [14]. A number of functional monomers has successfully been copolymerized for various applications; among them the immobilisation of biological compounds [15]. Even if (meth)acrylate monoliths were show to possess high efficiency towards particular biomolecules [14,16,17], it has never been shown that one particular polymer system is capable for high-resolution separation of the whole spectrum of biopolymers covering proteins, peptides, oligonucleotides as well as dsDNA, Nevertheless, it has to be noted that recently some attempts have been made for optimization of (meth)acrylate based monoliths for the separation of small molecules [18,19].
Styrene monoliths, mainly based upon copolymerization of styrene and divinylbenzene (PS/DVB), were finally shown to enable separation of the whole spectrum of biopolymers with unmatched resolution so far [20,21]. Those monoliths were commercialized by LC-Packings (Sunnyvale, Calif., USA). Styrene monoliths, however, seem to be inapplicable for the separation of small molecules, since no publication can be found on this topic.
The fact that the separation of small molecules on organic monoliths still is in its infancy [23] may be explained by the parameters presently employed for control of the porous properties of the polymer networks. The porogen composition [24], polymerization temperature [25] or initiator content [26] influence the distribution of macropores and thus the separation of analytes of high molecular weight (e.g. biomolecules) only. The possibility to control the fraction of mesopores, whose distribution is important for successful resolution of small molecules, is therefore conditional and insufficient.